Elsevier

Aquatic Toxicology

Volume 171, February 2016, Pages 30-36
Aquatic Toxicology

Hexavalent chromium is cytotoxic and genotoxic to American alligator cells

https://doi.org/10.1016/j.aquatox.2015.12.004Get rights and content

Highlights

  • Particulate Cr(VI) is cytotoxic and clastogenic to American alligator cells.

  • Soluble Cr(VI) is cytotoxic and clastogenic to American alligator cells.

  • Cr(VI) may be a risk factor for American alligator health.

Abstract

Metals are a common pollutant in the aquatic ecosystem. With global climate change, these levels are anticipated to rise as lower pH levels allow sediment bound metals to be released. The American alligator (Alligator mississippiensis) is an apex predator in the aquatic ecosystem and is considered a keystone species; as such it serves as a suitable monitor for localized pollution. One metal of increasing concern is hexavalent chromium (Cr(VI)). It is present in the aquatic environment and is a known human carcinogen and reproductive toxicant. We measured the cytotoxicity and genotoxicity of Cr(VI) in American alligator cells derived from scute tissue. We found that particulate and soluble Cr(VI) are both cytotoxic and genotoxic to alligator cells in a concentration-dependent manner. These data suggest that alligators may be used as a model for assessing the effects of environmental Cr(VI) contamination as well as for other metals of concern.

Introduction

The American alligator (Alligator mississippiensis) is a long lived, apex predator inhabiting primarily coastal areas of the southeastern US. Once listed as endangered due to overhunting, careful conservation efforts led to delisting in 1987. Localized populations of alligators are exposed to environmental contaminants through a variety of sources. Development in and near their ecosystem can lead to chemical and agricultural runoff. Since the alligator populations have successfully recovered after being listed as endangered in the 1970s, they can be used as a suitable monitor of environmental pollution (Delany et al., 1988).

Chromium (Cr) has recently been shown to be a metal of global concern (Wise et al., 2009). Hexavalent chromium (Cr(VI)) is a known human carcinogen and can damage DNA and impair reproduction and development (Al-Hamood et al., 1998, Bataineh et al., 1997, Chowdhury and Mitram, 1995, Holmes et al., 2008, IARC, 1990, Mancuso, 1997, Wise et al., 2008a, Wise et al., 2008b, Witmer et al., 1989). A few studies have measured Cr in alligators and reveal a concern. A study of alligators in South Carolina found a cluster of alligators had relatively high concentrations of Cr in liver tissue with several animals having levels over 30 μg/g (Campbell et al., 2010). Horai et al. (2014) showed that Cr accumulates in adult alligators based on comparisons between juvenile and adult alligator livers at 3 different sites in Florida. Interestingly of the three sites tested the Cr levels in adult alligators from Merritt Island National Wildlife Refuge (MINWR) were the highest and were 3 times higher than the other two sites suggesting localized pollution (Horai et al., 2014).

However, while these studies show alligators may be exposed to significant levels of Cr, no studies have considered potential adverse effects as a result of Cr exposure in alligators. In fact, consideration of the available literature shows Cr is poorly studied in aquatic reptiles and appears to be limited to two reports. One study, in green sea turtle cells, found Cr(VI) to be one of the most cytotoxic of four metals tested (Tan et al., 2010). The other considered hawksbill sea turtle cells and found both particulate and soluble Cr(VI) were cytotoxic and clastogenic (Wise et al., 2014).

The explanation for the lack of data is presumably due to the lack of access to experimental models of aquatic reptiles. Many species are endangered and protected. However, it is possible to gain important species-specific insights into potential toxicological impacts through aquatic reptile cell cultures. Accordingly, to begin developing a better understanding of pollution impacts on alligators and crocodilians in general, we investigated the cytotoxicity and genotoxicity of chromium in fibroblasts developed from American alligator scute tissue. Because the major health concern in the environment is the hexavalent form of chromium, and since particulate Cr(VI) is considered to be a more potent carcinogen than soluble Cr(VI) (IARC, 1990, Wise et al., 2008a, Wise et al., 2008b), we focused our study on particulate and soluble Cr(VI) compounds.

Section snippets

Materials

All plasticware was purchased from BD Falcon. Dulbecco’s Phosphate-Buffered Saline (DPBS), RPMI media with Glutagro was purchased from orning. Potassium chloride, demecolcine, lead chromate, and sodium chromate were purchased from Sigma/Aldrich. Crystal violet, methanol and acetic acid were purchased from JT Baker. Microscope slides were purchased from Thermo Scientific. Giemsa stain was manufactured by Rica Chemical Co. Fetal Bovine Serum (FBS), Gurr’s buffer, trypsin, penicillin-streptomycin

Cytotoxicity

There was a concentration-dependent decrease in alligator cell survival after both lead chromate and sodium chromate treatment compared to the untreated controls. Treatments of 0.1, 0.5, 1, and 5 μg/cm2 lead chromate for 24 h induced 93, 70, 60, and 1% relative survival, respectively (Fig. 1). Treatments of 0.5, 1, 2.5, and 5 μM sodium chromate induced 87, 78, 60, and 25% relative survival, respectively (Fig. 2). The estimated LC50s for lead chromate and sodium chromate were 2.1 μg/cm2 (95%

Discussion

Our data show particulate and soluble Cr(VI) are cytotoxic to alligator cells. Our study is the first to report cytotoxicity in alligator cells and one of only a few to report cytotoxicity in any reptilian species. Our data are consistent with previous cytotoxicity studies in hawksbill sea turtle, loggerhead sea turtle, and green sea turtle cells (Tan et al., 2010, Wang et al., 2013, Webb et al., 2014, Wise et al., 2014). Three of these studies considered metal cytotoxicity (Tan et al., 2010,

Acknowledgements

The authors would like to thank Matt Guillette for assistance in collecting alligator samples; Therry The and Christy Gianios, Jr. for technical support. Research reported in this publication was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number R01ES016893 (JPW). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional

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Present address: Wise Laboratory of Environmental and Genetic Toxicology, Department of Pharmacology and Toxicology, University of Louisville, 505 S. Prescott St, Louisville, KY 40292, USA.

2

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